Prothymosin alpha genes, pseudogenes, mRNA transcripts and proteins have been investigated with the following results: The gene family consists of six members, five of which can be classified as processed pseudogenes based on complete sequencing of the genes and their flanking regions. Although the pseudogenes possess consensus regulatory signals for transcription and translation and, in some cases, highly conserved open reading frames, none is expressed. The intron-containing gene gives rise to two transcripts by an unusual form of alternative splicing. As a result of nonconsensus splice acceptor selection at two adjacent GAG triplets, the second triplet, which would be expected to be exonic, is sequestered within the adjacent intron to generate the common form of prothymosin alpha mRNA. The rare form (about 10% of prothymosin alpha mature mRNA in all tissues examined) contains the triplet and encodes an identical protein made one residue longer by the insertion of a glutamic acid residue at position 39. These experiments have led to a refinement of the rules for splice acceptor selection. The mature protein is posttranslationally modified. The adduct has been identified and modified peptides have been isolated. Although experiments with synchronized human myeloma cells treated with antisense oligomers have shown that prothymosin alpha is required for cell division, modification of the protein(s) occurs throughout the cell cycle. Advances in understanding the function of prothymosin alpha in cell proliferation have been made as a consequence of: (1) devising a model system for use as an assay; (2) engineering a transgenic mouse; (3) fusing prothymosin alpha with a seven-amino acid epitope; and (4) constructing a CAT vector to investigate the interaction of the gene with c-Myc.